1. Field of the Invention
The present invention relates generally to neurobiology. More specifically, the present invention relates to molecular engineering of monoamine oxidases to determine those domains that play a role in the regulation of cellular neurotransmitters and vasoamines.
2. Description of the Related Art
The cytochrome P-450 superfamily of hemoproteins are extremely diverse in amino acid sequence, intron/exon organization, cellular localization, host organisms, substrates, and metabolic oxidation and reduction reactions. Common elements of the P-450s include a Soret absorption band at 450 nm associated with an obligatory heme cofactor, and conserved structural elements surrounding the heme in the active site. The xcex1-helix which traverses the active site of P-450cam and P-450BM-3 is also predicted in the primary and secondary structure of 52 species of membrane bound P-450 (16, 25). Although P-450BM-3 contains only 16% sequence identity to P-450cam and differs significantly from P-450cam in tertiary structure, redox partner preferences and substrate specificities, a region of high sequence homology is retained. In these P-450 enzymes, this conserved sequence resides within an extended xcex1-helix which traverses the active site.
Since this sequence is an integral part of the active site in P-450 enzymes, site-directed mutagenesis studies have been undertaken in numerous laboratories to identify specific residues that play a role in the active site (16). Although the specific role of several amino acid residues in this region are still being investigated, the residues which correspond to Asp251 and Thr252 of P-450cam are widely accepted as crucial for the activity of P-450 enzymes (17). For example, mutagenesis of those residues in P-450cam, P-450d or aromatase resulted in a dramatic decrease in activities (18). Furthermore, Gerber and Sligar (18) observed a significant change in the pH profile of P-450cam when Asp251 was changed to Asn (from parabolic to descending slope). They proposed a model in which the Asp251 and Thr252 residues of P-450cam provide a proton relay to protonate the heme-bound dioxygen prior to Oxe2x80x94O bond scission and water release.
The Asp251 residue of P-450cam has also been reported to play a role in substrate entry (20). Although a depression is observed at the surface of the protein that likely serves as the access channel for the substrate, the entry is blocked by the presence of a salt bridge (12). Deprez et al. (20) suggest that the salt bridge could control the opening of the structure. Specifically, Arg186, Lys178 (residues in P-450cam which reside outside of the active site xcex1-helix), and Asp251 were hypothesized to control substrate diffusion to the active site. Deprez et al. analyzed a D251N mutant enzyme by determining the effect of alteration of the ionic strength and dielectric constant of the medium on the substrate binding step. Substrate access was highly dependent on electrostatic interactions in the wild-type enzyme, but to a much lesser extent in the D251N mutant. Based upon these findings and the crystal structure resolved by Poulos et al. (12), Deprez et al. concluded that Asp-251 participates in a bifurcated salt bridge and plays a role in regulation of substrate diffusion to the active site.
Residue Thr252 of P-450cam is the most widely studied residue in this and other P-450 enzymes. Examination of the crystal structures of P-450cam suggested that Thr252 forms a hydrogen bond with the carbonyl oxygen of the conserved Gly248, causing an atypical kink in the helix to form the O2 binding pocket (21). However, this putative role for Thr252 was later eliminated when a mutant enzyme (T252A), which is incapable of hydrogen bonding with Gly248, was found to retain the same characteristic kink in the O2 binding pocket (22). Other studies support the conclusion that this conserved Thr of the cytochrome P-450 enzymes plays a role in substrate binding and specificity (23).
Monoamine oxidase A and B (MAO A and MAO B) are the major enzymes that catalyze the oxidative deamination of neuroactive and vasoactive amines in the central nervous system and peripheral tissues of mammals. These flavoenzymes are located in the outer mitochondrial membrane and are very similar in deduced amino acid sequence (1) and gene organization (2). MAO A and B can b e distinguished by differences in substrate preference and inhibitor specificity (3), tissue and cell distribution (4), and immunological properties (5).
Since the structure of MAO A or B has not been determined, attempts to identify active site domains have focused on substrate/inhibitor binding studies (6), symmetry modeling (7), FTIR spectroscopy (8), site-directed mutagenesis studies (9), analysis of MAO A/B chimeras (10), and expression of truncated polypeptides (11). Collectively, these studies provided increased insight into the functional regions of these enzymes, but the active site domain(s) and the specific residues that participate in the oxidative deamination of substrates have not been identified. Powell et al. (1), however, determined the amino acid sequence of bovine MAO A, and identified a short segment that exhibited high sequence identity to a region in cytochrome P-450 in rat.
The prior art is deficient in indentifying the active site of the monoamine oxidase (MAO) B enzyme and the effect that mutations to this site have on enzyme activity and the regulation of neuro- and vaso-amines. The present invention fulfills this long-standing need and desire in the art.
Monoamine oxidases A and B (MAO A and B) are the major enzymes in mammals that catalyze the oxidative deamination of neurotransmitters and peripheral vasoactive amines. Although these enzymes are among the most widely studied flavoproteins, their integral association with the outer mitochondrial membrane has precluded elucidation of their three-dimensional structure and identification of the active site domain(s). By comparing the primary sequence of MAO B to selected proteins of known structure, three amino acids (Phe423, Glu427, and Thr428) have been identified that constitute critical residues within the active site. The region in MAO B that contains these residues exhibits high sequence identity to a central active site helix in the cytochrome P-450 superfamily. Furthermore, this region, referred to herein as the conserved sequence, is predicted to have the same secondary structure as P-450cam and displays striking similarities in site-directed mutagenesis studies.
One object of the present invention is to provide isolated, genetically-engineered MAO B enzymes having at least one amino acid substitution for amino acids in the wildtype MAO B active site, where the wildtype amino acid is phenylalanine at position 423, glutamate at position 427 and threonine at position 428. The present invention additionally provides isolated DNAs that encode these genetically-engineered MAO B enzymes, and plasmids containing these DNAs along with regulatory elements necessary for expression of these DNAs in a cell.
Specific embodiments of this object of the present invention include where the wildtype amino acid is phenylalanine 423, the amino acid substitution is alanine; where the wildtype amino acid is glutamate 427, the amino acid substitution is glutamine; and where the wildtype amino acid is threonine 428, the amino acid substitution is serine or alanine.
An additional object of the present invention is to provide pharmaceutical compositions that interact with the active site of MAO B. Specific compositions include derivatives of active site components, such as FAD, 2xe2x80x2-deoxy FAD and 3xe2x80x2-deoxy FAD, and derivatives of mechanism-based inhibitors that belong to the acetylenic and cyclopropyl amine classes.
Additionally, an object of the present invention is to provide a method for regulating MAO B comprising the step of mutating an amino acid in the MAO B active site. Specifically, the amino acid to be mutated is selected from the wildtype amino acids Phe 423, Glu 427 and Thr 428.
Another object of the present invention is to provide a description of the active site of monoamine oxidase B, such that pharmaceutical compositions can be designed to interact with the active site. Specific embodiments of this object of the invention include 2xe2x80x2-deoxy FAD, 3xe2x80x2-deoxy FAD, and derivatives of deprenyl (phenylisopropyl-methylproinylamine) and trans-phenylcyclo propylamine. Molecular modeling is applied to determine which derivatives are most likely to interact with components in the active site of the enzyme.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments. These embodiments are given for the purpose of disclosure.